Many biological and chemical systems are composed of a large number of different interacting molecular species. The manner in which many of these molecules interact with each other determines the properties and functions of the particular system. For example, the function and properties of a particular biological system are due to the many and varied interactions that occur between the proteins, nucleic acids and other molecules that make up the system.
In order to understand how such complex systems function, it is necessary to define the individual interactions that occur between the different molecular species. A first step in defining these interactions is the identification of what molecular species are present in a system, and at what concentration they exist to exert their actions.
An improved understanding of the molecular species present in a complex system, and at what concentrations they exist, is also important in determining how some complex systems undergo a transition from one state to another state. For example, such considerations are important in understanding how the change from a normal state to a diseased state occurs for some cell types. An understanding of the identity and concentration of the molecular species present in a system is also important in terms of diagnosis and prognosis. For example, the transformation of a normal tissue to a pre-malignant tissue, and ultimately to a malignant one, may be able to be identified by an improved understanding of the presence and concentration of the molecular species present at any particular time in the cells of interest.
A powerful tool for the identification of the molecular species present in a complex mixture is the use of probe molecules that have the capacity to bind or interact with a particular molecule of interest. For example, antibodies may be used to identify specific antigens in complex mixtures of antigens. Naturally occurring ligands to a molecule (or engineered variants thereof) may be detectably labelled and used to identify their targets in complex mixtures of receptor molecules. Nucleic acids complementary to another specific nucleic acid may be used to identify and characterise the specific nucleic acid in a complex mixture of nucleic acids.
Accordingly, the generation of ligands with specificity to new or important target molecules is an important tool for research, diagnosis and treatment. However, the generation of new ligands to a specific target molecule is often problematic. In some cases, rational design of new ligands may be effective. In such instances a detailed understanding of the three dimensional structure of the relevant part of the target molecule is usually required. However, many target molecules (for example proteins) have complex structures, making the rational design of new ligands to the molecule difficult.
In some instances it is possible to identify new ligands to a target molecule without knowledge of the structure of the target molecule. In this case, the ability to identify new ligands is usually dependent upon the ability to generate a large number of molecules of different structure, a proportion of which may have the capacity to bind to a target molecule with useful affinity. For example, the generation of antibodies in vivo relies on such a principle. However, for the generation of antibodies specific to a particular target molecule it is usually necessary to first isolate the target antigen and/or screen a large number of monoclonal antibodies for binding to the target antigen. In addition, the use of antibodies as tools is often limited by the capacity to generate and isolate antibodies against specific types of target antigens, and the fact that the generation and testing of antibodies is a time consuming and labour intensive process.
Single stranded nucleic acids also have the capacity to form a multitude of different three dimensional structures. Indeed, single stranded nucleic acids may have a three dimensional structural diversity not unlike proteins. The three dimensional structure adopted by any one single stranded nucleic acid is dependent upon the primary sequence of nucleotides, and ultimately is the result of the numerous types of intra-molecular interactions that occur between atoms present in the molecule and inter-molecular interactions that occur between atoms present in the molecule and the surrounding solvent. The three dimensional structure will also depend upon the kinetics and thermodynamics of folding of any one structure.
Because single stranded nucleic acids have the capacity to form a multitude of different three dimensional structures, they may also be potential ligands to a large variety of different types of target molecules. Single stranded nucleic acids that have the capacity to bind to other target molecules are generally referred to as aptamers. In fact, given the structural diversity possible with single stranded nucleic acids; it may be possible to isolate a single stranded nucleic acid with a useful binding affinity to any molecule of interest.
In this regard, chemical synthesis of nucleic acids allows the generation of a pool of large numbers of single stranded nucleic acids of random nucleotide sequence. If the complexity of the pool of single stranded molecules generated by chemical synthesis is sufficient, it may be possible to isolate a unique nucleic acid ligand to any specific molecule. For example, SELEX (systematic evolution of ligands by exponential enrichment) is a technique that allows the isolation of specific nucleic acid ligands from a starting pool of candidate single stranded nucleic acids. By a process of reiterated steps of binding nucleic acids to a target molecule, isolation of the bound nucleic acids and subsequent amplification, nucleic acid ligands to a specific molecule may be quickly and easily identified.
However, a deficiency in the use of single stranded nucleic acid targets has been the inability to identify and use single stranded nucleic acid ligands to complex mixtures of molecules, as for example are present in cellular extracts. The large number of molecules present in the mixture, and the variety of interactions of varying affinity that are possible between molecules in the mixture and nucleic acid ligands, has made the identification and use of specific nucleic acid ligands to such mixtures problematic.
For example, the isolation of a specific nucleic acid ligand to a specific molecule by a process such as SELEX using purified, or even partially purified targets, does not necessarily result in a nucleic acid ligand that is effective in binding to the specific molecule when that molecule is present in a complex mixture of other potential target molecules. It would be advantageous to isolate nucleic acid ligands that can bind to specific molecules present in complex mixtures. It would also be advantageous to use such ligands to screen for differences in the concentration of specific target molecules between different sets of complex mixtures.
In addition, a further deficiency with the identification of nucleic acid ligands to complex mixtures has been the inability to readily produce a library of different nucleic acid ligands to the complex mixture. For example, it would ultimately be advantageous for many reasons to be able to readily isolate a unique nucleic acid ligand to every biologically significant molecule in a complex mixture.
To produce such a library of nucleic acid ligands by existing SELEX techniques would require the isolation of a specific target molecule present in the complex mixture and the independent isolation of a nucleic acid ligand to that specific molecule. In such a way, by repeating this process for each newly isolated molecule present in the complex mixture, a library of nucleic acid ligands to a number of different molecules in the complex mixture could be built up. However, not only is such a sequential manner of isolating nucleic acid ligands laborious and time consuming, the ligands so isolated may not be effective in binding to their specific target molecules, when those molecules are present in a complex mixture of other molecules.
The present invention relates to methods for the isolation of nucleic acid ligands that are capable of binding to target molecules present in complex mixtures.